Differentiation of Leishmania ( L .) infantum , Leishmania ( L .) amazonensis and Leishmania ( L .) mexicana Using Sequential qPCR Assays and High-Resolution Melt Analysis.

Leishmaniaprotozoa are the etiological agents of visceral, cutaneous and mucocutaneous leishmaniasis. In specific geographical regions, such as Latin America, severalLeishmaniaspecies are endemic and simultaneously present; therefore, a diagnostic method for species discrimination is warranted. In this attempt, many qPCR-based assays have been developed. Recently, we have shown thatL. (L.) infantumandL. (L.) amazonensiscan be distinguished through the comparison of the Cq values from two qPCR assays (qPCR-ML and qPCR-ama), designed to amplify kDNA minicircle subclasses more represented inL. (L.) infantumandL. (L.) amazonensis, respectively. This paper describes the application of this approach toL. (L.) mexicanaand introduces a new qPCR-ITS1 assay followed by high-resolution melt (HRM) analysis to differentiate this species fromL. (L.) amazonensis. We show thatL. (L.) mexicanacan be distinguished fromL. (L.) infantumusing the same approach we had previously validatedfor L. (L.) amazonensis. Moreover, it was also possible to reliably discriminateL. (L.) mexicanafromL. (L.) amazonensisby using qPCR-ITS1 followed by an HRM analysis. Therefore, a diagnostic algorithm based on sequential qPCR assays coupled with HRM analysis was established to identify/differentiateL. (L.) infantum,L. (L.) amazonensis,L. (L.) mexicanaandVianniasubgenus. These findings update and extend previous data published by our research group, providing an additional diagnostic tool in endemic areas with co-existing species.